Temporal Responses of Microbial Communities to Anaerobic Soil Disinfestation.

Anaerobic soil disinfestation (ASD) is an organic amendment-based management tool for controlling soil-borne plant diseases and is increasingly used in a variety of crops. ASD results in a marked decrease in soil redox potential and other physicochemical changes, and a turnover in the composition of the soil microbiome. Mechanisms of ASD-mediated pathogen control are not fully understood, but appear to depend on the carbon source used to initiate the process and involve a combination of biological (i.e., release of volatile organic compounds) and abiotic (i.e., lowered pH, release of metal ions) factors. In this study, we examined how the soil microbiome changes over time in response to ASD initiated with rice bran, tomato pomace, or red grape pomace as amendments using growth chamber mesocosms that replicate ASD-induced field soil redox conditions. Within 2 days, the soil microbiome rapidly shifted from a diverse assemblage of taxa to being dominated by members of the Firmicutes for all ASD treatments, whereas control mesocosms maintained diverse and more evenly distributed communities. Rice bran and tomato pomace amendments resulted in microbial communities with similar compositions and trajectories that were different from red grape pomace communities. Quantitative PCR showed nitrogenase gene abundances were higher in ASD communities and tended to increase over time, suggesting the potential for altering soil nitrogen availability. These results highlight the need for temporal and functional studies to understand how pathogen suppressive microbial communities assemble and function in ASD-treated soils.

Detection of Diazotrophy in the Acetylene-Fermenting Anaerobe Pelobacter sp. Strain SFB93.

Acetylene (C2H2) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C2H2 block assays of N2O reductase, and Pelobacter acetylenicus was shown to grow on C2H2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N2ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N2ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N2ase. Diazotrophic growth was observed under N2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N2ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days.IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants.

Degradation of Crude 4-MCHM (4-Methylcyclohexanemethanol) in Sediments from Elk River, West Virginia.

In January 2014, approximately 37 800 L of crude 4-methylcyclohexanemethanol (crude MCHM) spilled into the Elk River, West Virginia. To understand the long-term fate of 4-MCHM, we conducted experiments under environmentally relevant conditions to assess the potential for the 2 primary compounds in crude MCHM (1) to undergo biodegradation and (2) for sediments to serve as a long-term source of 4-MCHM. We developed a solid phase microextraction (SPME) method to quantify the cis- and trans-isomers of 4-MCHM. Autoclaved Elk River sediment slurries sorbed 17.5% of cis-4-MCHM and 31% of trans-4-MCHM from water during the 2-week experiment. Sterilized, impacted, spill-site sediment released minor amounts of cis- and up to 35 µg/L of trans-4-MCHM into water, indicating 4-MCHM was present in sediment collected 10 months post spill. In anoxic microcosms, 300 µg/L cis- and 150 µg/L trans-4-MCHM degraded to nondetectable levels in 8-13 days in both impacted and background sediments. Under aerobic conditions, 4-MCHM isomers degraded to nondetectable levels within 4 days. Microbial communities at impacted sites differed in composition compared to background samples, but communities from both sites shifted in response to crude MCHM amendments. Our results indicate that 4-MCHM is readily biodegradable under environmentally relevant conditions.

Evidence for high-temperature in situ nifH transcription in an alkaline hot spring of Lower Geyser Basin, Yellowstone National Park.

Genes encoding nitrogenase (nifH) were amplified from sediment and photosynthetic mat samples collected in the outflow channel of Mound Spring, an alkaline thermal feature in Yellowstone National Park. Results indicate the genetic capacity for nitrogen fixation over the entire range of temperatures sampled (57.2°C to 80.2°C). Amplification of environmental nifH transcripts revealed in situ expression of nifH genes at temperatures up to 72.7°C. However, we were unable to amplify transcripts of nifH at the higher-temperature locations (> 72.7°C). These results indicate that microbes at the highest temperature sites contain the genetic capacity to fix nitrogen, yet either do not express nifH or do so only transiently. Field measurements of nitrate and ammonium show fixed nitrogen limitation as temperature decreases along the outflow channel, suggesting nifH expression in response to the downstream decrease in bioavailable nitrogen. Nitrogen stable isotope values of Mound Spring sediment communities further support geochemical and genetic data. DNA and cDNA nifH amplicons form several unique phylogenetic clades, some of which appear to represent novel nifH sequences in both photosynthetic and chemosynthetic microbial communities. This is the first report of in situ nifH expression in strictly chemosynthetic zones of terrestrial (non-marine) hydrothermal systems, and sets a new upper temperature limit (72.7°C) for nitrogen fixation in alkaline, terrestrial hydrothermal environments.

Random forest analysis reveals taxa predictive of Prunus replant disease in peach root microbiomes.

Successive plantings of Prunus species produce suboptimal growth and yield in many California soils due to a poorly understood soilborne disease complex, Prunus replant disease (PRD). We explored the hypothesis that PRD is mediated by microbial taxa in roots of Nemaguard peach, a rootstock for almond and other stone fruits. In a greenhouse bioassay, portions of 10 replant soils were treated with fumigation or pasteurization or left untreated as a control before being planted with peach seedlings. Ten weeks after planting, seedlings were considered PRD-affected if their top fresh weights in the control were significantly reduced, compared to the weights in pasteurization and fumigation treatments; plants with equivalent top weights in all treatments were considered to be non-affected. The roots were washed from the soil, frozen, extracted for total DNA, and used for metabarcoding of rRNA gene amplicons from bacteria, fungi, and oomycetes. High-throughput amplicon sequencing revealed that root microbial community shifts resulted from preplant treatments, and specific taxa were associated with PRD induction among controls. Random forest (RF) analysis discriminated effectively between PRD-affected and non-affected root communities. Among the 30 RF top-ranked amplicon sequence variant (ASV) predictors, 26 were bacteria, two were oomycetes, and two were fungi. Among them, only Streptomyces scabiei, Steroidobacter denitrificans, Streptomyces bobili, and Pythium mamillatum had root abundances ≥5% that were either associated positively (former two ASVs) or negatively (latter two) with PRD. Thus, our findings were consistent with microbial mediation of PRD in roots and suggested taxa that may be involved in the mediation.

Effects of whole-orchard recycling on nitrate leaching potential in almond production systems.

Inefficient nitrogen (N) fertilization and irrigation have led to unhealthy nitrate levels in groundwater bodies of agricultural areas in California. Simultaneously, high commodity prices and drought have encouraged perennial crop growers to turnover less-productive orchards, providing opportunities to recycle tree biomass in situ and to use high-carbon (C) residues to conserve soil and water resources. Although climate change adaptation and mitigation benefits of high-C soil amendments have been shown, uncertainties remain regarding the benefits and trade-offs of this practice for N cycling and retention. We used established almond [Prunus dulcis (Mill.) D. A. Webb] orchard trials on Hanford fine sandy loam with short-term and long-term biomass recycling legacies to better understand the changes in N dynamics and retention capacity associated with this practice. In a soil column experiment, labeled N fertilizer was added and traced into various N pools, including microbial biomass and inorganic fractions in soil and leachate. Shifts in microbial communities were characterized using the abundance of key N cycling functional genes regulating nitrification and denitrification processes. Our findings showed that, in the short term, biomass recycling led to N immobilization within the orchard biomass incorporation depth zone (0-15 cm) without impacts on N leaching potential. However, this practice drastically reduced nitrate leaching potential by 52%, 10 yr after biomass incorporation without an increase in N immobilization. Although the timing of these potential benefits as a function of microbial population and C and N biogeochemical cycles still needs to be clarified, our results highlight the potential of this practice to meaningfully mitigate nitrate discharges into groundwater while conserving soil resources.

What makes a natural clay antibacterial?

Natural clays have been used in ancient and modern medicine, but the mechanism(s) that make certain clays lethal against bacterial pathogens has not been identified. We have compared the depositional environments, mineralogies, and chemistries of clays that exhibit antibacterial effects on a broad spectrum of human pathogens including antibiotic resistant strains. Natural antibacterial clays contain nanoscale (<200 nm), illite-smectite and reduced iron phases. The role of clay minerals in the bactericidal process is to buffer the aqueous pH and oxidation state to conditions that promote Fe(2+) solubility. Chemical analyses of E. coli killed by aqueous leachates of an antibacterial clay show that intracellular concentrations of Fe and P are elevated relative to controls. Phosphorus uptake by the cells supports a regulatory role of polyphosphate or phospholipids in controlling Fe(2+). Fenton reaction products can degrade critical cell components, but we deduce that extracellular processes do not cause cell death. Rather, Fe(2+) overwhelms outer membrane regulatory proteins and is oxidized when it enters the cell, precipitating Fe(3+) and producing lethal hydroxyl radicals.

Characterization of soils conducive and non-conducive to Prunus replant disease.

Successive orchard plantings of almond and other Prunus species exhibit reduced growth and yield in many California soils. This phenomenon, known as Prunus replant disease (PRD), can be prevented by preplant soil fumigation or anaerobic soil disinfestation, but its etiology is poorly understood and its incidence and severity are hard to predict. We report here on relationships among physicochemical variables, microbial community structure, and PRD induction in 25 diverse replant soils from California. In a greenhouse bioassay, soil was considered to be "PRD-inducing" when growth of peach seedlings in it was significantly increased by preplant fumigation and pasteurization, compared to an untreated control. PRD was induced in 18 of the 25 soils, and PRD severity correlated positively with soil exchangeable-K, pH, %clay, total %N, and electrical conductivity. The structure of bacterial, fungal, and oomycete communities differed significantly between the PRD-inducing and non-inducing soils, based on PERMANOVA of Bray Curtis dissimilarities. Bacterial class MB-A2-108 of phylum Actinobacteria had high relative abundances among PRD-inducing soils, while Bacteroidia were relatively abundant among non-inducing soils. Among fungi, many ASVs classified only to kingdom level were relatively abundant among PRD-inducing soils whereas ASVs of Trichoderma were relatively abundant among non-inducing soils. Random forest classification effectively discriminated between PRD-inducing and non-inducing soils, revealing many bacterial ASVs with high explanatory values. Random forest regression effectively accounted for PRD severity, with soil exchangeable-K and pH having high predictive value. Our work revealed several biotic and abiotic variables worthy of further examination in PRD etiology.

Fungal and bacterial communities of 'Pinot noir' must: effects of vintage, growing region, climate, and basic must chemistry.

BACKGROUND: The geographic and temporal distributions of bacterial and fungal populations are poorly understood within the same wine grape cultivar. In this work, we describe the microbial composition from 'Pinot noir' must with respect to vintage, growing region, climate, and must chemistry across the states of California and Oregon, USA. MATERIALS AND METHODS: We sampled 'Pinot noir' clone 667 clusters from 15 vineyards existing in a latitudinal gradient spanning nearly 1,200 km in California and Oregon for two vintages (2016 and 2017). Regions included five American Viticultural Areas (AVA). In order from southern California to Oregon, these AVAs were Santa Barbara, Monterey, Sonoma, Mendocino, and Willamette Valley. Uninoculated grape musts were subjected to 16S rRNA gene and ITS-1 amplicon sequencing to assess composition of microbial communities. We also measured grape maturity metrics. Finally, to describe regions by precipitation and growing degree days, we queried the Parameter-elevation Regressions on Independent Slopes Model (PRISM) spatial climate dataset. RESULTS: Most of the dominant bacterial taxa in must samples were in the family Enterobacteriaceae, notably the lactic acid bacteria or the acetic acid bacteria groups, but some, like the betaproteobacterial genus Massilia, belonged to groups not commonly found in grape musts. Fungal communities were dominated by Hanseniaspora uvarum (Saccharomycetaceae). We detected relationships between covariates (e.g., vintage, precipitation during the growing season, pH, titratable acidity, and total soluble solids) and bacterial genera Gluconobacter and Tatumella in the family Enterobacteraceae, Sphingomonas (Sphingomonodaceae), Lactobacillus (Lactobacillaceae), and Massilia (Oxalobacteraceae), as well as fungal genera in Hanseniaspora, Kazachstania, Lachancea, Torulaspora in the family Saccharomycetaceae, as well as Alternaria (Pleosporaceae), Erysiphe (Erysiphaceae), and Udeniomyces (Cystofilobasidiaceae). Fungal community distances were significantly correlated with geographic distances, but this was not observed for bacterial communities. Climate varied across regions and vintages, with growing season precipitation ranging from 11 mm to 285 mm and growing degree days ranging from 1,245 to 1,846. DISCUSSION: We determined that (1) bacterial beta diversity is structured by growing season precipitation, (2) fungal beta diversity reflects growing season precipitation and growing degree days, and (3) microbial differential abundances of specific genera vary with vintage, growing season precipitation, and fruit maturity metrics. Further, the correlation between fungal community dissimilarities and geographic distance suggests dispersal limitation and the vineyard as a source for abundant fungal taxa. Contrasting this observation, the lack of correlation between bacterial community dissimilarity and geographic distance suggests that environmental filtering is shaping these communities.

Genomic adaptations in information processing underpin trophic strategy in a whole-ecosystem nutrient enrichment experiment.

Several universal genomic traits affect trade-offs in the capacity, cost, and efficiency of the biochemical information processing that underpins metabolism and reproduction. We analyzed the role of these traits in mediating the responses of a planktonic microbial community to nutrient enrichment in an oligotrophic, phosphorus-deficient pond in Cuatro Ciénegas, Mexico. This is one of the first whole-ecosystem experiments to involve replicated metagenomic assessment. Mean bacterial genome size, GC content, total number of tRNA genes, total number of rRNA genes, and codon usage bias in ribosomal protein sequences were all higher in the fertilized treatment, as predicted on the basis of the assumption that oligotrophy favors lower information-processing costs whereas copiotrophy favors higher processing rates. Contrasting changes in trait variances also suggested differences between traits in mediating assembly under copiotrophic versus oligotrophic conditions. Trade-offs in information-processing traits are apparently sufficiently pronounced to play a role in community assembly because the major components of metabolism-information, energy, and nutrient requirements-are fine-tuned to an organism's growth and trophic strategy.

Complete Genome Sequences of Brenneria rubrifaciens Strain 6D370 and Brenneria nigrifluens Strain ATCC 13028, Causative Agents of Bark Cankers in Walnut.

Brenneria rubrifaciens and Brenneria nigrifluens, respectively, cause deep and shallow bark canker disease in walnut. B. rubrifaciens exhibits quorum sensing-controlled virulence and rubrifacine pigment production. The complete genome sequences of these species will be useful for studying the role of genes regulated by quorum sensing, including pathways mediating pathogenesis.

Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707.

Molecular analysis of the amo gene cluster in Nitrosococcus oceani revealed that it consists of five genes, instead of the three known genes, amoCAB. The two additional genes, orf1 and orf5, were introduced as amoR and amoD, respectively. Putative functions of the AmoR and AmoD proteins are discussed.

Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter.

The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet approximately 21 kb of a approximately 28-kb "autotrophic" island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b(561), and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter "subcore" genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris. Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter.

Draft Genome Sequence of Agrobacterium tumefaciens Biovar 1 Strain 186, Isolated from Walnut.

Agrobacterium tumefaciens biovar 1 strain 186 was isolated from a walnut tree expressing crown gall symptoms. The draft genome sequence of this strain harbored genes for crown gall formation and will be useful for understanding its virulence on Paradox, the predominant hybrid rootstock used for the cultivation of English walnut in California.

Complete Genome Sequences of Two Acetylene-Fermenting Pelobacter acetylenicus Strains.

Acetylene fermentation is a rare metabolism that was serendipitously discovered during C2H2-block assays of N2O reductase. Here, we report the genome sequences of two type strains of acetylene-fermenting Pelobacter acetylenicus, the freshwater bacterium DSM 3246 and the estuarine bacterium DSM 3247.

Complete Genome Sequence of the Acetylene-Fermenting Pelobacter sp. Strain SFB93.

Acetylene fermentation is a rare metabolism that was previously reported as being unique to Pelobacter acetylenicus Here, we report the genome sequence of Pelobacter sp. strain SFB93, an acetylene-fermenting bacterium isolated from sediments collected in San Francisco Bay, CA.

Nutrient Stoichiometry Shapes Microbial Community Structure in an Evaporitic Shallow Pond.

Nutrient availability and ratios can play an important role in shaping microbial communities of freshwater ecosystems. The Cuatro Ciénegas Basin (CCB) in Mexico is a desert oasis where, perhaps paradoxically, high microbial diversity coincides with extreme oligotrophy. To better understand the effects of nutrients on microbial communities in CCB, a mesocosm experiment was implemented in a stoichiometrically imbalanced pond, Lagunita, which has an average TN:TP ratio of 122 (atomic). The experiment had four treatments, each with five spatial replicates - unamended controls and three fertilization treatments with different nitrogen:phosphorus (N:P) regimes (P only, N:P = 16 and N:P = 75 by atoms). In the water column, quantitative PCR of the 16S rRNA gene indicated that P enrichment alone favored proliferation of bacterial taxa with high rRNA gene copy number, consistent with a previously hypothesized but untested connection between rRNA gene copy number and P requirement. Bacterial and microbial eukaryotic community structure was investigated by pyrosequencing of 16S and 18S rRNA genes from the planktonic and surficial sediment samples. Nutrient enrichment shifted the composition of the planktonic community in a treatment-specific manner and promoted the growth of previously rare bacterial taxa at the expense of the more abundant, potentially endemic, taxa. The eukaryotic community was highly enriched with phototrophic populations in the fertilized treatment. The sediment microbial community exhibited high beta diversity among replicates within treatments, which obscured any changes due to fertilization. Overall, these results showed that nutrient stoichiometry can be an important factor in shaping microbial community structure.

Response of a stoichiometrically imbalanced ecosystem to manipulation of nutrient supplies and ratios.

Cuatro Ciénegas Basin (CCB) is a desert ecosystem that hosts a large diversity of water bodies. Many surface waters in this basin have imbalanced nitrogen (N) to phosphorus (P) stoichiometry (total N:P > 100 by atoms), where P is likely to be a limiting nutrient. To investigate the effects of nutrient stoichiometry on planktonic and sediment ecosystem components and processes, we conducted a replicated in situ mesocosm experiment in Lagunita, a shallow pond located in the southwest region of the basin. Inorganic N and P were periodically added to mesocosms under three different N:P regimes (P only, N:P = 16 and N:P = 75) while the control mesocosms were left unamended. After three weeks of fertilization, more than two thirds of the applied P was immobilized into seston or sediment. The rapid uptake of P significantly decreased biomass C:P and N:P ratios, supporting the hypothesis that Lagunita is P-limited. Meanwhile, simultaneous N and P enrichment significantly enhanced planktonic growth, increasing total planktonic biomass by more than 2-fold compared to the unenriched control. With up to 76% of added N sequestered into the seston, it is suspected that the Lagunita microbial community also experienced strong N-limitation. However, when N and P were applied at N:P = 75, the microbes remained in a P-limitation state as in the untreated control. Two weeks after the last fertilizer application, seston C:P and N:P ratios returned to initial levels but chlorophyll a and seston C concentrations remained elevated. Additionally, no P release from the sediment was observed in the fertilized mesocosms. Overall, this study provides evidence that Lagunita is highly sensitive to nutrient perturbation because the biota is primarily P-limited and experiences a secondary N-limitation despite its high TN:TP ratio. This study serves as a strong basis to justify the need for protection of CCB ecosystems and other low-nutrient microbe-dominated systems from anthropogenic inputs of both N and P.

Draft Genome Sequence of Microvirga sp. Strain BSC39, Isolated from Biological Soil Crust of Moab, Utah.

Microvirga sp. BSC39 was isolated from a biological soil crust near Moab, Utah. The strain appears to be capable of chemotaxis and exopolysaccharide synthesis for biofilm adhesion. The BSC39 genome contains iron siderophore uptake and hydrolysis enzymes; however, it lacks siderophore synthesis pathways, suggesting the uptake of siderophores produced by neighboring microbes.

Draft Genome Sequence of Bacillus sp. Strain BSC154, Isolated from Biological Soil Crust of Moab, Utah.

Bacillus sp. BSC154 was isolated from a biological soil crust near Moab, Utah. The strain appears to be capable of chemotaxis and biofilm production. The BSC154 genome contains iron siderophore production, nitrate reduction, mixed acid-butanediol fermentation, and assimilatory and dissimilatory sulfate metabolism pathways.